In Drosophila, two groups of genes, the Polycomb group (PcG) and the Trithorax group (TrxG) are important for inheritance of the silenced and the active chromatin state, respectively. Regulatory elements called Polycomb group response elements (PREs) are cis-acting DNA sequences required for the recruitment of chromatin-modifying PcG protein complexes. Recently it has been suggested that TrxG proteins act through either the same or overlapping cis-acting sequences as PcG proteins. Our group is working on understanding how PcG and TrxG proteins get recruited to the DNA. Polycomb group response elements (PREs) are DNA elements through which the Polycomb group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many different proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. Our goal is to define all of the DNA binding proteins required for the activity of this PRE, and to ask whether all PREs are made up of binding sites for the same proteins. Disruption of any of seven different binding sites within this 181-bp PRE disrupts PRE function. The proteins that bind five of these sites are known: Pho/Phol, GAF/Psq, Zeste, Dsp1, and members of the Sp1/KLF family. Our lab identified Pho and Phol and has been studying their function. Pho and Phol are related proteins that bind the same sequence and have some redundant activities. Pho and Phol are required for PcG protein function and for binding of PcG proteins to the DNA. pho, phol double mutant animals have a PcG phenotype clearly indicating they play a central important role in PcG repression. More recently we have been studying the Sp1/KLF family proteins in Drosophila, to try to determine whether a family member plays a central role in PcG protein silencing. This family of proteins encodes transcription factors and has been extensively studied in mammals. There are 20 Sp1/KLF family members in mammals. In Drosophila there are 9 Sp1/KLF family members, and nine of them bind to a site in the 181-bp PRE we study. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila. During the past year we have been working on determining which of the Sp1/KLF family members in Drosophila may be involved in PcG function. We made antibodies to the three most likely candidates and identified one that we named Spps (Sp1-factor for pairing-sensitive silencing) that binds to PREs. We made a mutation in Spps, and find that it is necessary for pairing-sensitive silencing, an activity dependent on PcG activity. We expected that Spps mutants would display a PcG phenotype, but this was not the case. We suspect that, like Pho and Pho-like, there is another Sp1/Klf family member that acts redundantly with Spps to silence PcG targets. We are currently working to identify the activity of the other Sp1/KLF family members. Another important aspect of our work is to ask whether all PREs are alike. In this regard we characterized PREs from the Drosophila invected gene as well as an additional PRE from the engrailed gene. Our work suggests that there is not one way to make a PRE. For example, it was suggested in the literature that two Pho/Phol binding sites are required for PRE function. Our laboratory found that this is true for some PREs, but in the 181-bp PRE, only one Pho/Phol binding site is necessary for PRE function. This difference has implications on the mechanism of PcG protein complex recruitment to the DNA. The PRE we study is from the Drosophila engrailed gene. engrailed encodes a homeodomain protein that plays an important role in the development of many different parts of the embryo including the formation of the segments, nervous system, head, and gut. It also plays a very important role in the development of the adult, specifying the posterior compartment of each imaginal disk. Accordingly, engrailed is expressed in a very specific and complex manner in the developing organism. The 181-bp engrailed PRE we have been studying is located near the engrailed promoter from 576 to 395bp upstream of the transcription start site. We are interested in determining the role of this PRE in the control of engrailed expression. One of the first things we learned in our studies is that this PRE is redundant with other flanking PREs in the endogenous engrailed gene. There is another strong PRE located from -1100 to -1500 bp and probably other weak PREs nearby. In fact, when we examined the location of Ph and Pho proteins on engrailed DNA by chromatin immunoprecipation (ChIP), we found that they are bound to a 2.5 kb region extending from the engrailed promoter to about 2.5kb upstream. Therefore, it is perhaps not too surprising that a 500bp deletion that includes the 181-bp PRE and flanking sequence did not lead to ectopic engrailed expression. The remaining PREs were apparently sufficient to recruit PcG proteins. However, what was surprising to us was that loss of this DNA lead to a loss of function phenotype, suggesting that this DNA must also play a positive role in the expression of engrailed. Our experiments suggest that there are multiple positive elements either overlapping with or coincident with the PREs. Our work also suggests that PREs can mediate looping, either with distant PREs or with distant enhancers. Our work shows that PREs can either activate or repress transcription, dependent on the context. This is an important result consistent with the idea that PREs are also the site of action of Trithorax group proteins. Finally, we have recently found that sequences required for promoter-enhancer communication are located within or near the engrailed PREs. The regulatory sequences for the engrailed gene extend over a 70 kb region. Our lab has used reporter constructs to find sequences important for expression in stripes, the nervous system, the head, etc. There are discrete regulatory elements located throughout the 70kb region. We also find at least 7 additional PREs located throughout the region. PcG protein complexes have been shown in vitro to bring together DNA fragments, and it is possible that they cause looping in vivo. We are interested in learning whether the additional PREs are involved in mediating interactions between distant enhancers and the engrailed promoter.